Abortion is the ending of pregnancy by the removal or forcing out of a fetus or embryo from the uterus before it is able to survive on its own. An abortion can occur spontaneously, in which case it is often called a miscarriage (Grimes and Stuart, 2010). Spontaneous abortion may occur for many reasons, not all of which can be identified. Some of these causes include genetic, uterine, or hormonal abnormalities, reproductive tract infections, and tissue rejection. Abortion caused by invasive prenatal diagnosis such as chorionic villus sampling and amniocentesis is rare (about 1%) (Agarwal and Alfirevic, 2012).
Spontaneous abortion is usually seen before the 12th week of pregnancy (Qumsiyeh et al., 2000). The overall risk of spontaneous abortion after 15 weeks is low (about 0.6%) for chromosomally and structurally normal fetuses, but varies according to maternal age and ethnicity (Wyatt et al., 2005). Chromosomal abnormalities are found in more than half of the embryos miscarried in the first 13 weeks (Kajii et al., 2013). Other causes of spontaneous abortion include immunological abnormalities, endocrine-metabolic disorders, and congenital uterine abnormalities (Kelten et al., 2010). Studies attempting to evaluate apoptosis in normal and abnormal pregnancies have been performed; the studies used proliferation markers such as Ki-67 and proliferating cell nuclear antigen as well as many apoptosis-related genes or proteins, mainly p53 and Bcl-2 and others (e.g. Fas/FasL system and Bax), in fetomaternal tissues (Kelten et al., 2010).
Apoptosis is an ordered and orchestrated cellular process that occurs in physiological and pathological conditions. It is also one of the most studied topics among cell biologists (Wong, 2011). Understanding apoptosis in disease conditions is very important as it not only provides insights into the pathogenesis of a disease but may also leave clues on how the disease can be treated (Bauer and Hefand, 2006). The Bcl-2 family of proteins is comprised of proapoptotic and antiapoptotic proteins that play a pivotal role in the regulation of apoptosis, especially through the intrinsic pathway, as they reside upstream of irreversible cellular damage and act mainly at the mitochondria level (Weng, et al., 2008). When there is disruption in the balance of antiapoptotic and proapoptotic members of the Bcl-2 family, the result is dysregulated apoptosis in the affected cells. This can be due to an overexpression of one or more antiapoptotic proteins or an underexpression of one or more proapoptotic proteins or a combination of both (Czabotar et al., 2014). Several studies in humans have reported the presence of endometrial apoptotic cells appearing mainly at the beginning of the secretory phase during the receptive period, becoming more numerous during the late secretory phase, and finally peaking during the menstrual phase (Castro et al., 2002). Definition of apoptotic changes during the early pregnancy may therefore contribute to the prevention of unwanted abortions and recurrent loss of pregnancy. Disturbances in the regulation of cellular proliferation and apoptosis can directly or indirectly lead to fetal growth retardation and loss of pregnancy. The rate of apoptosis in third trimester placentas with growth retardation has been reported to be much higher than that in normal third trimester placentas (Qumsiyeh et al., 2000).
The aim of this study was to analyze the agents that play a role in the apoptotic process by evaluating the relations between a proliferation marker (Ki-67) and an apoptosis-related marker (Bcl-2) immunohistochemically in abortus material, specifically fetal trophoblasts and maternal deciduas.
Materials and methods
The material of this work consisted of curettage material of 30 cases of spontaneous abortion together with 10 cases of normal placenta. They were collected from the surgical files of the Histopathology Departments of Al-Azhar University Hospital (Al-Hussein and Al-Zahraa University Hospitals) during the period 2012–2014. The clinical and obstetric data were retrieved from the files of the patients.
For histopathological examination, 5-µm-thick sections were prepared from each tissue paraffin block and stained with hematoxylin and eosin for confirmation of the diagnosis. This step was performed by Mohamed Y. Ali, Eman M. Ahmed, Marwa A. Elkholy, and Mohamed S. Abd Elwahed.
For immunohistochemical staining, two sections were cut from each case on positively charged slides and subjected to immunohistochemical staining using the streptavidin–biotin alkaline phosphate methods.
The primary antibodies used for immunohistochemical staining (with clone, manufacture, dilution, incubation period, and positive control) were as follows: Ki-67 [mouse monoclonal prediluted (ready to use), Clone MIB1, N1633; Dako cytomation, Glostrup, Denmark], and BCL-2 (monoclonal antibody; Dako Cytomation, Glostrup, Denmark, at 1 : 200 dilution).
Positive control for two antibodies was sections of tonsils. Negative controls were prepared by omitting the primary antibody under identical test condition.
The sections were placed in an oven at 50°C for 30 min and then the sections were deparaffinized in xylene, rehydrated in graded alcohol dilution, washed in PBS, incubated with 0.3% hydrogen peroxide to block endogenous peroxidase activity, washed in PBS again, and boiled in citrate buffer solution (pH 6.0) using a microwave for 10 min at 60°C for antigen retrieval. After cooling at room temperature, the sections were incubated with primary antibody overnight in a humidified chamber and rinsed with PBS. The sections were then incubated for 30 min at 37°C with biotinylated secondary antibody and streptavidin conjugated to horseradish peroxidase. After three rinses with PBS, the sections were incubated with diaminobenzidine substrate and then rinsed with distilled water and counterstained with hematoxylin. This step was evaluated independently and jointly by Mohamed Y. Ali, Eman M. Ahmed, Marwa A. Elkholy and Mohamed S. Abd Elwahed.
Interpretation of immunohistochemical staining
When immunohistochemical staining was evaluated, the whole section was scanned at the ×10 magnification of the microscope in each case. A magnification of ×40 was used to calculate the rate of positive staining. Nuclear staining was considered for Ki-67, and cell membrane and cytoplasmic staining for Bcl-2.
Immunoexpression of Ki-67 in villous cytotrophoblasts, syncytiotrophoblasts, and decidual stromal cells was assessed as follows: 0, no stained cells; +, 25% or greater positive cells; ++, 26–50% positive cells; and +++, more than 50% positive cells (Khooei et al., 2013).
Bcl-2 expression of villous cytotrophoblasts, syncytiotrophoblasts, and decidual stromal cells was evaluated according to the intensity of staining and the proportion of stained villous cells and was scored as follows: −, no staining; +, weak staining; ++, moderate staining; and +++, intense staining (Rath et al., 2011).
Ki-67 immunoreactivity was assessed in the nuclei of the villous cytotrophoblasts and decidual stromal cells, whereas syncytotrophoblasts showed absence of immunostaining (Table 1). Strong Ki-67 expression was detected in the cytotrophoblastic nuclei of 10 of the 30 cases of abortion (33.3%), whereas it was expressed in the nuclei of eight out of the 10 cases (80%) of the control group and it was expressed in the nuclei of decidual stromal cells of 10 of the 30 cases of abortion (33.3%) compared with nine of 10 cases of the control group (90%) (Fig. 1). The difference between the two groups was statistically significant (P=0.048 and 0.014, respectively).
Bcl-2 immunoreactivity was detected in the cytoplasm of villous syncytiotrophoblasts and decidual stromal cells of cases of abortion and maternal placenta but the cytotrophoblasts showed negative immunostaining (Table 2). Bcl-2 was expressed in the cytoplasm of syncytiotrophoblast cells of the abortion group as strong in only three of 30 cases (10%), whereas it was expressed in the syncytiotrophoblast cells of the control group as strong in six out of 10 cases (60%).
Concerning the decidual stromal cells, Bcl-2 expression was intense in only three of 30 (10%) cases of the abortion group, whereas it was expressed intensely in four of 10 (40%) cases of the control group.
There was a significant difference in Bcl-2 immunoreactivity in syncytiotrophoblasts and stromal decidual cells between the abortion group and the control group (P=0.003 and 0.054, respectively). The statistical analysis demonstrated that the expression of Bcl-2 was significantly downregulated in spontaneous abortion (P=0.003) as compared with maternal placenta, and the differences was statistically significant (Table 2) (Fig. 2).
Relationship between Ki-67 and Bcl-2 expression in the abortion group
Of the 30 abortion specimens examined, 20 (66.7%) cases showed Ki-67 expression (0 and +) and displayed a loss of Bcl-2 expression (Table 3). Statistical analysis showed a significant association between nuclear Ki-67 and cytoplasmic Bcl-2 protein expression in villous trophoblastic cells (P=0.000) (Table 3). No significant associations were detected between Ki-67 and Bcl-2 expression in decidual stromal cells (P=0.6).
Studies on cellular proliferation in placental tissue have focused on the trophoblastic cells as they are in direct contact with maternal blood and vital to maintain a successful pregnancy. Adequate proliferation is required for both trophoblastic cells and villous stromal cells and for blood vessels for the maturation and branching of villi (Kelten et al., 2010). Unlike cytotrophoblast, which is the trophoblastic stem cell, syncytiotrophoblast is the terminally differentiated cell that produces most of the placental hormones and regulates the diffusion of oxygen, CO2, and other nutrients between the mother and the fetus (Shih and Kurman, 2001). Khooei et al. (2013) and Kelten et al. (2010) studied proliferation markers in the human placenta during the normal course of pregnancy and reported highest Ki-67 expression in villous cytotrophoblasts in the first trimester of pregnancy, which was lower in term placenta. However, Ki-67 expression has not been reported in syncytiotrophoblasts during pregnancy.
In this study, the immunoreactivity for Ki-67 in abortions and normal placentas was largely confined to cytotrophoblasts. This pattern is in accordance with that described in the previous studies of Khooei et al. (2013), which identified the cytotrophoblast as the active germinative zone based on the results obtained using total organ DNA analysis, flow cytometry, morphometric analysis, and studies of proliferating cell nuclear antigen.
In the current work, the proliferation rate for both fetal villi and maternal decidua evaluated by Ki-67 expression was significantly lower in the abortion group than in the control group. Strong Ki-67 nuclear staining of cytotrophoblasts and decidual stromal cells was detected in 10/30 cases of abortion, whereas it was detected in 8/10 and 9/10 syncytiotrophoblasts and decidual stromal cells, respectively, of the control group.
The significant decrease in the proliferative rate evaluated with Ki-67, found in the villous cytotrophoblasts and maternal decidua in the abortion group in this work, may explain the changes in placenta with inadequate development, which produce a lower level of hCG that decreases the progesterone support of the decidua and leads to abortion in the early period.
Apoptosis has been reported in the human placenta under both normal and pathological conditions and Bcl-2 was the first of its family to be discovered (Sgarbosa et al., 2006). Several molecules are associated with the induction and prevention of apoptosis. Bcl-2 is one such molecule whose expression is considered as an antiapoptotic factor that is responsible for inhibiting apoptosis (Murakoshi et al., 2003).
In this study, Bcl-2 was expressed in the cytoplasm of villous syncytotrophoblasts and stromal cells of both the abortion and control groups. However, the cytotrophoblasts showed negative immunostaining. There was a decrease in BCL-2 expression in abortus material as compared with normal placenta. On the basis of these results, it is suggested that the increased apoptotic activity in the decidual tissue that plays an important role in the nutrition of the developing embryo, protection from maternal immunological responses, and the regulation of the uterine stromal invasion by trophoblasts could be responsible for the termination of pregnancy in spontaneous abortion. It is also proposed that the increased apoptosis in decidual tissue in spontaneous abortion could trigger apoptotic activity in the syncytiotrophoblasts as well and thus could prevent embryonic development. However, Halperin et al. (2000) did not find any significant difference between normal pregnancies (first and second trimester) and missed abortion materials as regards the apoptotic cell percentage using flow cytometry and the propidium iodide staining method. They emphasized that these various changes may be due to some type of necrosis consisting of structural changes such as minimal changes in the nuclear morphology, swelling of organelles and cytoplasm, and separation of plasma membrane instead of apoptosis. This difference in Bcl-2 expression in many reports is thought to be due to the use of different procedures for Bcl-2 evaluation.
Ishihara et al. (2000) evaluated cytotrophoblast and syncytiotrophoblast Bcl-2 levels in the human placenta and found Bcl-2 expression to be at the lowest level during the early weeks of pregnancy (week 4–5), with levels increasing towards the term and mainly in the syncytiotrophoblastic cell cytoplasm. They reported that Bcl-2 expression in syncytiotrophoblasts that increases towards term may be important in maintaining pregnancy and protecting placental integrity by preventing loss of these cells.
Kelten et al. (2010) found that Bcl-2 expression was lower in the spontaneous and recurrent abortion groups compared with the normal placenta group, although this was not statistically significant. This decrease in Bcl-2 expression was found in the syncytiotrophoblastic cells lining the villi in fetal tissues.
The current study showed a significant association between nuclear Ki-67 and cytoplasmic Bcl-2 protein expression in villous trophoblastic cells (P=0.000), but no significant associations were detected between Ki-67 and Bcl-2 expression in decidual stromal cells (P=0.6). These findings suggest that decreased proliferation of cytotrophoblastic cells, indicated by low Ki-67 expression, associated with increased apoptosis of syncytiotrophoblasts, indicated by low Bcl-2 protein expression in abortion, can be important causes in the pathogenesis of spontaneous abortion.
Conflicts of interest
There are no conflicts of interest.
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